In integrated and miniaturized biosensor devices, also called lab-on-chip devices, the biosensor is part of a microfluidic platform that allows for a rapid and automated in-flow detection of the analyte. To ensure optimal sensitivity, a site-selective coupling of the analyte-recognizing molecules or receptors on the sensor area is required.
Different surface chemistries have been tried, mainly self-assembled monolayers of organosilanes, to ensure a covalent binding of the analyte-recognizing molecules to various biosensor devices. For this purpose, silanes with a functional group that is reactive towards the analyte-recognizing molecules, e.g. antibodies, has been used, such as epoxy-silanes. However, these reactive functional groups are prone to hydrolysis and their stability is limited upon storage especially in H2O-containing atmospheres. An alternative, more preferred and widely used approach is to use less reactive groups in combination with a linker group. An example of this approach has been described by J. Ryken et al. (sensors and actuators B 200 (2014) 167-172) and consists in using in a first step, a cross-linker to activate the silane so that it can, in a second step, react with the analyte-recognizing molecule. This is done using a two-step protocol where first the biosensor substrate is activated using the cross-linker followed by the conjugation of the analyte-recognizing molecule in a second reaction.
In order to immobilize the analyte-recognizing molecules on full wafer scale in a cost-effective way, non-contact microarray printing technologies (‘spotting’) to generate arrays of microdroplets on the area of the biosensor transducer are mostly suited. In order to enable the spotting, both the substrate and the solutions used to spot the analyte-recognizing molecules should be sufficiently stable. Additionally, the two-step approach involves spot-on-spot deposition with an intermediate washing step which is not easy to implement in practice, especially not in a high-throughput environment. There is therefore a need in the art for methods for immobilizing analyte-recognizing molecule on surfaces that overcome or improve on one or more of the above mentioned drawbacks of the prior art.